Project Summary Posttranslational modification of proteins by ubiquitin (Ub) is a regulatory mechanism that controls nearly all aspects eukaryotic cell biology. Ubiquitination alters properties of target proteins such as stability, subcellular localization, intermolecular interactions, and activity and thereby regulates processes such as cell cycle control, DNA repair, signal transduction, and immunity. The relevance of Ub signaling to human health is underscored by the fact that its dysregulation is implicated in pathologies such as cancer, neurological disorders, cardiovascular disease, and immune disorders and that it is a validated target for therapeutic intervention in cancer with FDA-approved medications extending the lives of multiple myeloma patients. Ub signaling requires the sequential interactions and activities of three enzymes, E1, E2, and E3, which act in tandem to conjugate Ub to target proteins. Humans harbor two Ub E1s, Uba1 and Uba6, that catalyze Ub activation and thioester transfer to distinct repertoires of tens of E2s. While Uba1 is fully dedicated to Ub activation, Uba6 is highly unusual in that it is also capable of activating FAT10 (a Ub-like protein involved in mitotic progression and immunity), and subsequently transferring it to a highly Uba6-specific E2, UBE2Z. Maintenance of the integrity of Ub signaling is essential, yet mechanisms underlying Uba6 promiscuity for Ub and FAT10, as well as the molecular rules governing specificity/promiscuity in E1/E2 interactions remain poorly understood. After E1-E2 thioester transfer, E2~Ub intermediates interact with distinct repertoires of hundreds of E3 ligases grouped into three families that catalyze ubiquitination of target proteins as a single molecule or as polymeric chains linked together by specific lysine residues on Ub. Because it is a major determinant of the functional outcome of ubiquitination, control of the type of polyUb chains assembled on substrate proteins is essential and for reactions catalyzed by RING E3s, polyUb chain specificity is largely determined by the E2 with which they function. Despite this fundamental importance, the molecular mechanisms governing specificity in catalysis of most polyUb linkage types remain unknown. Through use of structural, biochemical/biophysical, and cell-based approaches, this proposal aims to discover: 1) the structural basis for substrate recognition and catalytic activities of human Ub E1 enzymes 2) the catalytic mechanism of E1-E2 thioester transfer and molecular rules governing specificity/promiscuity in Ub E1/E2 interactions, and 3) mechanisms by which specific types of polyUb chains are catalyzed by E2/RING E3 pairs. Ub signaling is a target for therapeutic intervention in cancer and other human pathologies and the deeper understanding of how E1, E2, and E3 work together to control essential cellular processes that will result from the proposed studies could provide a platform for the development of novel small molecule therapeutics.